Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition

Abstract

The Saccharomyces cerevisiae DBR1 gene encodes a 2-5 phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5 end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2-5 phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.Saccharomyces cerevisiae Ty1 is a long terminal repeat (LTR) retroelement with a life cycle similar to that of retroviruses (2). Like retroviruses, Ty1 replicates via reverse transcription of an RNA intermediate, subsequently integrating element cDNA into the host genome. Retroviruses and LTR retrotransposons rely on their hosts to provide most of the factors necessary for propagation. We identified dbr1 mutants in a screen to identify host cell genes required for Ty1 transposition (15). The dbr1 mutant had been identified previously in a similar screen by others (6). Although dbr1 cells produce wild-type levels of Ty1 proteins, they accumulate Ty1 cDNA at a slower rate than do wild-type cells, suggesting that Dbr1p plays a role in Ty1 reverse transcription or cDNA stability (15).In its cellular role, Dbr1p acts at the end of the mRNA splicing process, cleaving the 2Ј-5Ј linkage at the branch point of intron RNA lariats released after exon ligation, converting them to linear RNAs that are rapidly degraded (6, 23). In a dbr1 mutant, intron RNAs accumulate as lariats (6). The uncleaved lariat branch point blocks the progression of 3Ј exonucleases, and there is no 5Ј end available on which a 5Ј exonuclease can act. DBR1 is highly conserved, and homologues have been cloned from Schizosaccharomyces pombe, S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (6,17,18,24). Deletion of DBR1 has no significant effect on the growth rate of S. cerevisiae, but...

LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq

“…This intriguing finding raises the possibility of a feedback from floating lariats to the splicing machinery to prevent further lariat accumulation. A reduction in splicing efficiency was also observed in the ACT1 locus of debranchingdeficient budding yeast cells, and the accumulated ACT1 intron may slow down later stages of the splicing reaction (Salem et al 2003). Decreased recycling of spliceosomal RNAs and proteins, which might not be released from stable lariats, could lead to the decreased splicing efficiency (Arenas and Abelson 1997;Martin et al 2002;Hilleren and Parker 2003).…”

Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown

“…Cheng and Menees (8) provided evidence that during cDNA synthesis the Ty1 RNA genome contains a 2=-5= branch characteristic of an RNA lariat, although these data remain controversial (10). The location of this branch connecting the 5= end of the genome to the 3= nucleotide of the U3 region suggested that it may play a role during Ty1 cDNA synthesis by facilitating the transfer of nascent minus-strand cDNA from the 5= end of the Ty1 RNA template to the 3= R region (11). However, this possibility was subsequently challenged (10).…”

Ty1 reverse transcriptase does not read through the proposed 2′,5′-branched retrotransposition intermediate in vitro

“…The most likely candidate mechanism remains the use of linear 59+39 Ty1 RNAs as the template (Fig. 1A), with the two RNAs held together noncovalently by the CYC base-pairing interaction (Cristofari et al 2002) (Salem et al 2003). Alternatively, Dbr1p could be required in vivo to remove a primer that initiated cDNA synthesis via its 29-hydroxyl group, or Dbr1p could have an indirect role via modulating deoxyribonucleotide concentrations (Lauermann et al 1995;Karst et al 2000).…”

“…This postulated branch read-through ability by Ty1 RT may seem unlikely, given that mapping of 29,59-branch sites by RTs-which halt at the branch sites-is well known from early work on RNA splicing (Domdey et al 1984;Rodriguez et al 1984;Zeitlin and Efstratiadis 1984). However, both Cheng and Menees (2004) as well as Perlman and Boeke (2004) suggested that read-through of the proposed Ty1 branch by Ty1 RT is plausible on the basis of several experiments with unrelated RTs, which showed that in at least some instances RTs can eventually read through either a 29,59-branch site (Vogel et al 1997;Tuschl et al 1998;Vogel and Borner 2002;Salem et al 2003) or a linear 29-59 linkage (Lorsch et al 1995). This situation brings into sharp focus a key question: Is Ty1 RT actually capable of reading through the proposed Ty1 RNA branch site?…”

“…Mutant dbr1 cells lacking Dbr1p nevertheless can produce substantial Ty1 cDNA (Karst et al 2000;Griffith et al 2003;Mou et al 2006) and have a substantial Ty1 transposition frequency when compared with wild-type cells (Salem et al 2003). As noted by Perlman and Boeke (2004) in their commentary on the original paper (Cheng and Menees 2004), if the hypothesis of an obligatory Ty1 branched RNA intermediate is correct, then it must be the case that ''the Ty1 RT may occasionally yield full-length cDNA by reading through the 29,59-branch (perhaps without strand transfer).''…”